Journal of Chromatography B (v.803, #1)
FM iii: Full-title Page (ii).
Peptide separation and analysis by Harald John; Ludger Ständker (1-2).
Peptidomics by Geert Baggerman; Peter Verleyen; Elke Clynen; Jurgen Huybrechts; Arnold De Loof; Liliane Schoofs (3-16).
Peptides occur in the whole animal kingdom, from the least evolved phyla with a very simple nervous system (coelenterates) to the highest vertebrates and are involved in most, if not all, physiological processes in animals.Knowing the amino acid sequence of peptide hormones or neurotransmitters is important since this allows for synthesis of large quantities of peptides to perform further functional analysis. Immunocytochemistry, radioimmunoassays (RIA), enzyme-linked immunosorbant assays (ELISA) and mass spectrometry can then provide information on the temporal and spatial distribution and quantification of the (neuro)peptide. Ever since the 1970s, a wealth of peptides has been discovered and investigated and this flow seems to be far from over. This is partially due to the use of new approaches mainly based on chromatographical purifications as well as molecular biological techniques.Surprisingly, peptides have so far been neglected in most proteomic studies. The finalization of the genome projects has opened new opportunities for rapid identification and functional analysis of (neuro)peptides as well. In analogy with the proteomics technology, where all proteins expressed in a cell or tissue are analyzed, the peptidomic approach aims at the simultaneous visualization and identification of the whole peptidome of a cell or tissue, i.e. all expressed peptides with their post-translational modifications (PTMs). This technology provides us with a fast and efficient tool to analyze the peptides from any tissue. This paper reviews the approaches that have been used so far to achieve this.
Keywords: Reviews; Peptide profiling; Proteomics; Neuropeptides;
Differential polypeptide display: the search for the elusive target by Stefan Wittke; Thorsten Kaiser; Harald Mischak (17-26).
Proteomics, as a tool to identify proteins in biological samples, is gaining rapidly importance in the postgenomic era. Here we discuss the current and potential role of different techniques in the field of proteomics such as two-dimensional gel electrophoresis off-line coupled to MALDI-MS (2D-PAGE-MALDI-MS), high performance liquid chromatography mass spectrometry (HPLC-MS), surface enhanced laser desorption/ionization mass spectrometry (SELDI-MS) and a newly developed technique, capillary electrophoresis mass spectrometry (CE-MS). The developments of the last years are presented discussed.
Keywords: Reviews; Proteomics; Data processing; Polypeptides;
Methodological advances in the discovery of protein and peptide disease markers by Rainer Bischoff; Theo M. Luider (27-40).
The quest for biomarkers has seen a renaissance due to the application of newly developed separation methodologies and advances in biomolecular mass spectrometry. It can be argued that each disease influences the physiology of an organism and that these changes should be measurable. Many diagnostic and therapeutic decisions are supported by measurable biochemical or cellular changes in plasma, serum or urine but it is unquestionable that there is a great lack in better markers for early disease detection and prevention. In this review we cover recent developments in the areas of separation science, sample preparation and mass spectrometry as applied to biomarker discovery. We focus, in particular, on the use of LC-MS and SELDI-TOF-MS as two approaches that have seen an upswing in recent years. While validation of newly discovered biomarkers or biomarker patterns and their introduction into diagnostic practice will be a long process, it is our believe that many future diagnostic tests will be based on markers discovered through novel profiling technologies as those outlined in this article.
Keywords: Reviews; Biomarker; Proteomics;
Bioactive peptides from marine sources: pharmacological properties and isolation procedures by Abel Aneiros; Anoland Garateix (41-53).
Marine organisms represent a valuable source of new compounds. The biodiversity of the marine environment and the associated chemical diversity constitute a practically unlimited resource of new active substances in the field of the development of bioactive products.In this paper, the molecular diversity of different marine peptides is described as well as information about their biological properties and mechanisms of action is provided. Moreover, a short review about isolation procedures of selected bioactive marine peptides is offered.Novel peptides from sponges, ascidians, mollusks, sea anemones and seaweeds are presented in association with their pharmacological properties and obtainment methods.
Keywords: Reviews; Isolation; Marine sources; Peptides, bioactive;
Proteomics of the venom from the Amazonian scorpion Tityus cambridgei and the role of prolines on mass spectrometry analysis of toxins by Cesar V.F Batista; Luis del Pozo; Fernando Z Zamudio; Sandra Contreras; Baltazar Becerril; Enzo Wanke; Lourival D Possani (55-66).
Scorpion venom are complex mixtures of peptides, known to cause impairment of ion-channel function in biological membranes. This report describes the separation of approximately 60 different components by high performance liquid chromatography and the characterization by Edman degradation and mass spectrometry of 26 peptides from the soluble venom of the Amazonian scorpion Tityus cambridgei. One of these peptides, named Tc48a, was fully characterized. It contains 65 amino acid residues, the C-terminal residue is amidated and it affects Na+-channels with a K d of about 82 nM. Furthermore, this report shows the thermo-instability of scorpion toxins subjected to electron spray ionization-mass spectrometry (ESI-MS). When a proline residue is located near the N-terminal region of the toxin, not stabilized by disulfide bridges, artificial components are generated by the mass spectrometer conditions, due to the cleavage of the peptide bond at the proline positions. This phenomenon was confirmed by using four model proteins (variable regions of immunoglobulins) studied by ESI-MS and matrix assisted laser desorption ionization–time of flight (MALDI–TOF)/MS.
Keywords: Proteomics; Tityus cambridgei; Toxins; Proline;
Structure–function study of a chlorotoxin-chimer and its activity on Kv1.3 channels by Isabelle Huys; Etienne Waelkens; Jan Tytgat (67-73).
Chlorotoxin has been isolated from the venom of the scorpion Leiurus quinquestriatus and characterized as a 4.1 kDa peptide, containing a lysine at position 27 that is also present in many Kv-blocking toxins. Because chlorotoxin shows no affinity for Kv-channels, we intended to design, express and purify a chlorotoxin-chimer, containing the active binding site (β-sheet) of a very potent Kv1-channel blocking peptide, agitoxin 2, by mutating three original residues in the chlorotoxin molecule. Several derivatives of the chimer, gradually missing one additional amino acid residue at the N-terminal side of the peptide, were produced and identified chromatographically. In contrast to chlorotoxin, these chimer derivatives are capable of blocking cloned Kv1-channels.
Keywords: Structure–function study; Kv1.3 channels; Chlorotoxin-chimer;
Recombinant production, purification and biochemical characterization of domain 6 of LEKTI: a temporary Kazal-type-related serine proteinase inhibitor by Peter Kreutzmann; Axel Schulz; Ludger Ständker; Wolf-Georg Forssmann; Hans-Jürgen Mägert (75-81).
Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is a 15-domain serine proteinase inhibitor which is of pathophysiological relevance for skin diseases and atopy. Domains 2 and 15 of LEKTI contain six cysteine residues and match the Kazal-type inhibitor motif almost exactly. The other 13 domains seem to be Kazal-type derived but lack the cysteines in positions 3 and 6 usually conserved within this family of inhibitors. Here, we report the recombinant production and comprehensive biochemical characterization of the 7.7 kDa LEKTI domain 6 (LD-6). Testing a selected number of different serine proteinases, we show that both native and recombinant LD-6 exhibit a significant but temporary inhibitory activity on trypsin. Furthermore, the relation of LEKTI domain 6 to Kazal-type inhibitors is confirmed by determining its disulfide bond pattern (1–4/2–3) and its P1 site located after the second Cys residue of LD-6. The established strategy for the recombinant production of LEKTI domain 6 will enable further investigation of its mode of action and its physiological role.
Keywords: Recombinant production; Purification; Characterization; LEKTI, domain 6; Serine proteinase inhibitor;
Quantitative analysis of [Dmt1]DALDA in ovine plasma by capillary liquid chromatography-nanospray ion-trap mass spectrometry by Haibao Wan; Edward S. Umstot; Hazel H. Szeto; Peter W. Schiller; Dominic M. Desiderio (83-90).
The synthetic opioid peptide analog Dmt-D-Arg-Phe-Lys-NH2 ([Dmt1]DALDA; Dmt=2′,6′-dimethyltyrosine) is a highly potent and selective mu opioid-receptor agonist. A very sensitive and robust capillary liquid chromatography/nanospray ion-trap (IT) mass spectrometry method has been developed to quantify [Dmt1]DALDA in ovine plasma, using deuterated [Dmt1]DALDA as the internal standard. The standard MS/MS spectra of d0- and d5-[Dmt1]DALDA were obtained, and the collision energy was experimentally optimized to 25%. The product ion [M+2H-NH3]2+ (m/z 312.2) was used to identify and to quantify the synthetic opioid peptide analog in ovine plasma samples. The MS/MS detection sensitivity for [Dmt1]DALDA was 625 amol. A calibration curve was constructed, and quantitative analysis was performed on a series of ovine plasma samples.
Keywords: [Dmt1]DALDA; Opioid peptide;
Simultaneous determination of incretin hormones and their truncated forms from human plasma by immunoprecipitation and liquid chromatography–mass spectrometry by Raik Wolf; Torsten Hoffmann; Fred Rosche; Hans-Ulrich Demuth (91-99).
The incretins, glucose-dependent insulinotropic peptide (GIP1–42) and glucagon-like peptide 1 (GLP-17–36), are involved in regulation of gastric emptying, glucose homeostasis, body fat regulation and the glucose-induced insulin secretion from the endocrine pancreas. After release in the circulation both peptides are rapidly degraded by the exopeptidase dipeptidyl peptidase IV (DP IV) to the inactive polypeptides GIP3–42 and GLP-19–36. In vivo stabilization of the active incretins by orally available DP IV-inhibitors is now widely accepted as a new therapeutic approach in antidiabetic treatment. In order to demonstrate the pharmacodynamic effect of DP IV-inhibitors, it is necessary to measure the plasma levels of active and inactive forms of GIP and GLP-1. We previously described an immunoprecipitation method as sample preparation and concentration in combination with a LC–MS analysis for determination of active and inactive GIP. We could improve the efficiency and suitability of this method by reduction of the necessary sample volume to 1.0 ml and simultaneous measurement of GIP1–42, GIP3–42 and GLP-17–36, GLP-19–36, without loss of sensitivity. An LOQ of approximately 5 and 11 pmol/l was maintained for GIP and GLP-1, respectively.
Keywords: Immunoprecipitation; Incretin hormones;
Optimization of reversed-phase microcapillary liquid chromatography for quantitative proteomics by Hookeun Lee; Eugene C Yi; Bo Wen; Timothy P Reily; Lance Pohl; Sidney Nelson; Ruedi Aebersold; David R Goodlett (101-110).
Currently, the field of shotgun proteomics relies primarily on the separation of peptides by reversed-phase microcapillary chromatography (RP-μLC) combined with either electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) and tandem mass spectrometry (MS/MS) for protein identification as well as quantification. For this purpose we herein describe construction of a RP-μLC–ESI column-emitter along with optimized μLC conditions for using the device to quantify pair-wise changes in protein expression via the isotope coded affinity tag (ICAT™) method that also maximize peak capacity. These optimized RP-μLC parameters required a balance be reached between the disparate needs of quantification which requires good peak shape and identification (i.e. proteome coverage) of proteins via peptide collision induced dissociation (CID) which requires peak capacity be maximized. A complex biological sample from a study of murine acetaminophen toxicity in hepatocyes was chosen for method development because of the high level complexity, but the biological results are not the focus of this manuscript.
Keywords: Optimization; Quantitative proteomics;
Identification and analysis of phosphopeptides by Michael J Chalmers; Walter Kolch; Mark R Emmett; Alan G Marshall; Harald Mischak (111-120).
Reversible phosphorylation of serine, threonine and tyrosine residues in proteins is one of the key events in signal transduction. To understand the process of signal transduction on a molecular level, it is imperative to identify phosphorylation sites in proteins. In this review, we offer an overview of the different methods/technologies currently available to identify protein phosphorylation sites.
Keywords: Reviews; Phosphorylation; Post-translational modification; Phosphopeptides;
Automated multi-dimensional liquid chromatography: sample preparation and identification of peptides from human blood filtrate by Egidijus Machtejevas; Harald John; Knut Wagner; Ludger Ständker; György Marko-Varga; Wolf-Georg Forssmann; Rainer Bischoff; Klaus K. Unger (121-130).
A comprehensive on-line sample clean-up with an integrated two-dimensional HPLC system was developed for the analysis of natural peptides. Samples comprised of endogenous peptides with molecular weights up to 20 kDa were generated from human hemofiltrate (HF) obtained from patients with chronic renal failure. The (poly-)peptides were separated using novel silica-based restricted access materials with strong cation-exchange functionalities (SCX-RAM). The size-selective sample fractionation step is followed by cation-exchange chromatography as the first dimension. The subsequent second dimension of separation is based on hydrophobic interaction using four parallel short reversed-phase (RP) columns implemented via a fully automated column switching technique. More than 1000 peaks were resolved within the total analysis time of 96 min. Substances of selected peaks were sampled to analyse their molecular weights by off-line MALDI-TOF mass spectrometry and to determine their amino acid sequence by Edman degradation. The potential for comprehensive peptide mapping and identification is demonstrated.
Keywords: Sample preparation; Identification; Human blood filtrate; Peptides;
Protein identification by liquid chromatography–mass spectrometry using retention time prediction by Magnus Palmblad; Margareta Ramström; Christopher G Bailey; Sandra L McCutchen-Maloney; Jonas Bergquist; Loreen C Zeller (131-135).
Liquid chromatography has been coupled with mass spectrometry to improve the dynamic range and to reduce the complexity of sample introduced to the mass spectrometer at any given time. The chromatographic separation also provides information on the analytes, such as peptides in enzymatic digests of proteins; information that can be used when identifying the proteins by peptide mass fingerprinting. This paper discusses a recently introduced method based on retention time prediction to extract information from chromatographic separations and the applications of this method to protein identification in organisms with small and large genomes.
Keywords: Protein identification; Retention time prediction;
Evaluation and comparison of tailor-made stationary phases based on spherical silica-based beads for capillary electrochromatography via peptide separation analysis by M.I. Huber; T.P. Hennessy; Dieter Lubda; K.K. Unger (137-147).
Small cyclic peptides have been employed to elucidate the performance of novel sorbents as stationary phases in capillary electrochromatography (CEC). In this paper chain length dependencies for ordinary liquid chromatographic sorbents are reported together with findings acquired on beads specifically designed to suit CEC. The latter, tailor-made, spherical, porous silica exhibits a distinguished surface modification to meet the criteria anticipated to enhance performance profiles in CEC. With well-characterised peptides resembling the analytes, probing of the CEC system in a systematic manner (predominantly via the organic modifier content of the background electrolyte (BE)) reveals insight into the complex interplay occurring in such analytical systems at the molecular and sub-molecular level in particular upon various modes of interaction.
Keywords: Stationary phases; Electrochromatography; Peptide separation analysis;
Optimization of capillary coating by hydroxyethyl methacrylate for capillary zone electrophoresis of proteins by Anke Feldmann; Ute Claußnitzer; Matthias Otto (149-157).
This work describes further improvements of coating fused silica capillaries with 2-hydroxyethyl methacrylate (HEMA) by atom transfer radical polymerization (ATRP). First, endcapping with a sterically less bulky silanyl reagent reduces the electrosmotic flow (EOF) by 25% in addition to the 40% EOF reduction caused by HEMA coating compared to a bare fused silica capillary. An additional hydrolysis step was introduced into the preparation of HEMA coated capillaries and leads to better reproducible migration times. The influence of the solvent during ATRP and the resulting polymer coating was investigated by replacement of DMF with water or water–methanol mixtures. The quality of the optimized coating was characterized by protein separations at pH 3. HEMA coated capillaries reveal up to 746 000 plates. The polyvinyl alcohol (PVA) coated capillary provides only half of this efficiency. A long-term test at pH 9 shows good stability of the HEMA coated capillaries in basic medium. Also the numbers of plates in this medium was about 30% higher than for separations with the PVA capillary. In addition, the phosphate buffer was replaced by a volatile ammonium acetate buffer for later use with mass spectrometry (MS).
Keywords: Optimization; Capillary coating; Hydroxyethyl methacrylate; Proteins;
General method allowing the use of 100% aqueous loading conditions in reversed-phase liquid chromatography by Sylvia Winkel Pettersson; Börje S Persson; Mats Nyström (159-165).
Reversed-phase HPLC purification of peptides, using n-alkyl modified spherical silica, has become a widely used technique within the pharmaceutical industry. One drawback of these materials is the necessity of having at least 5% organic modifier in the mobile phase, in order to avoid de-wetting of the porous stationary phase.For some preparative reversed-phase separations, it is an advantage if the feed solution can be loaded onto the column under 100% aqueous conditions.This study describes the use of post-column pressure control to avoid de-wetting of regular reversed-phase stationary phases when operated under 100% aqueous conditions. The applicability of post-column pressure control as a means of maintaining the column fully wetted is demonstrated with various buffers and with packing materials having different alkyl-chain lengths.Two peptides, insulin and oxytocin, in overloaded quantities, were loaded under 100% aqueous conditions onto a regular C8 column, and then eluted by a acetonitrile gradient following standard procedures. The retention volume and the peak shape showed that the separation was satisfactory, and proved that post-column pressure control can be used to overcome wettability problems, which are otherwise often observed for reversed-phase packing materials with high ligand density.
Keywords: Wettability; Aqueous loading conditions;
Mass spectrometric strategy for primary structure determination of N-terminally blocked peptides by Ren-Huai Huang; Da-Cheng Wang (167-172).
The mass spectrometric strategy including three steps is presented for primary structure determination of the N-terminally blocked peptides. First, the C-terminal sequencing is performed by using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry coupled with carboxypeptidase Y digestion. Then, the peptide is cleaved according to the obtained C-terminal sequence information and the resulting peptides are identified by mass spectrometry and Edman degradation after fractionation by reverse-phase chromatography. Finally, the N-terminal fragment is sequenced by tandem mass spectrometry. The strategy was successfully applied to the sequence determination of two novel N-terminally blocked peptides named EAFP1 and EAFP2.
Keywords: Primary structure determination; Peptides, N-terminally blocked;